Cavaloc Y, Popielarz M, Fuchs J P, Gattoni R, Stévenin J
Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
EMBO J. 1994 Jun 1;13(11):2639-49. doi: 10.1002/j.1460-2075.1994.tb06554.x.
By adopting a monoclonal antibody approach, we have identified a novel splicing factor of 35 kDa which we have termed 9G8. The isolation and characterization of cDNA clones indicate that 9G8 is a novel member of the serine/arginine (SR) splicing factor family because it includes an N-terminal RNA binding domain (RBD) and a C-terminal SR domain. The RNA binding domain of 9G8 is highly homologous to those of the SRp20 and RBP1 factors (79-71% identity), but the homology is less pronounced in the cases of SF2/ASF and SC35/PR264 (45-37% identity). Compared with the other SR splicing factors, 9G8 presents some specific sequence features because it contains an RRSRSXSX consensus sequence repeated six times in the SR domain, and a CCHC motif in its median region, similar to the zinc knuckle found in the SLU7 splicing factor in yeast. Complete immunodepletion of 9G8 from a nuclear extract, which is accompanied by a substantial depletion of other SR factors, results in a loss of splicing activity. We show that a recombinant 9G8 protein, expressed using a baculovirus vector and excluding other SR factors, rescues the splicing activity of a 9G8-depleted nuclear extract and an S100 cytoplasmic fraction. This indicates that 9G8 plays a crucial role in splicing, similar to that of the other SR splicing factors. This similarity was confirmed by the fact that purified human SC35 also rescues the 9G8-depleted extract. The identification of the 9G8 factor enlarges the essential family of SR splicing factors, whose members have also been proposed to play key roles in alternative splicing.
通过采用单克隆抗体方法，我们鉴定出一种35 kDa的新型剪接因子，我们将其命名为9G8。cDNA克隆的分离和表征表明，9G8是丝氨酸/精氨酸（SR）剪接因子家族的一个新成员，因为它包含一个N端RNA结合结构域（RBD）和一个C端SR结构域。9G8的RNA结合结构域与SRp20和RBP1因子的RNA结合结构域高度同源（同一性为79 - 71%），但在SF2/ASF和SC35/PR264的情况下，同源性不太明显（同一性为45 - 37%）。与其他SR剪接因子相比，9G8呈现出一些特定的序列特征，因为它在SR结构域中包含一个重复六次的RRSRSXSX共有序列，并且在其中间区域有一个CCHC基序，类似于酵母中SLU7剪接因子中发现的锌指结构。从核提取物中完全免疫去除9G8，同时伴随着其他SR因子的大量减少，导致剪接活性丧失。我们表明，使用杆状病毒载体表达且不包含其他SR因子的重组9G8蛋白可挽救9G8缺失的核提取物和S100细胞质组分的剪接活性。这表明9G8在剪接中起关键作用，类似于其他SR剪接因子。纯化的人SC35也能挽救9G8缺失的提取物这一事实证实了这种相似性。9G8因子的鉴定扩大了SR剪接因子这一重要家族，其成员也被认为在可变剪接中起关键作用。
